Genetic analysis and molecular basis of G6PD deficiency among malaria patients in Thailand: implications for safe use of 8-aminoquinolines.

BACKGROUND: It was hypothesized that glucose-6-phosphate dehydrogenase (G6PD) deficiency confers a protective effect against malaria infection, however, safety concerns have been raised regarding haemolytic toxicity caused by radical cure with 8-aminoquinolines in G6PD-deficient individuals. Malaria elimination and control are also complicated by the high prevalence of G6PD deficiency in malaria-endemic areas. Hence, accurate identification of G6PD deficiency is required to identify those who are eligible for malaria treatment using 8-aminoquinolines. METHODS: The prevalence of G6PD deficiency among 408 Thai participants diagnosed with malaria by microscopy (71), and malaria-negative controls (337), was assessed using a phenotypic test based on water-soluble tetrazolium salts. High-resolution melting (HRM) curve analysis was developed from a previous study to enable the detection of 15 common missense, synonymous and intronic G6PD mutations in Asian populations. The identified mutations were subjected to biochemical and structural characterisation to understand the molecular mechanisms underlying enzyme deficiency. RESULTS: Based on phenotypic testing, the prevalence of G6PD deficiency (< 30% activity) was 6.13% (25/408) and intermediate deficiency (30-70% activity) was found in 15.20% (62/408) of participants. Several G6PD genotypes with newly discovered double missense variants were identified by HRM assays, including G6PD Gaohe + Viangchan, G6PD Valladolid + Viangchan and G6PD Canton + Viangchan. A significantly high frequency of synonymous (c.1311C>T) and intronic (c.1365-13T>C and c.486-34delT) mutations was detected with intermediate to normal enzyme activity. The double missense mutations were less catalytically active than their corresponding single missense mutations, resulting in severe enzyme deficiency. While the mutations had a minor effect on binding affinity, structural instability was a key contributor to the enzyme deficiency observed in G6PD-deficient individuals. CONCLUSIONS: With varying degrees of enzyme deficiency, G6PD genotyping can be used as a complement to phenotypic screening to identify those who are eligible for 8-aminoquinolines. The information gained from this study could be useful for management and treatment of malaria, as well as for the prevention of unanticipated reactions to certain medications and foods in the studied population.

A computational study of structural analysis of Class I human glucose-6-phosphate dehydrogenase (G6PD) variants: Elaborating the correlation to chronic non-spherocytic hemolytic anemia (CNSHA).

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect that affects more than 500 million people worldwide. Individuals affected with G6PD deficiency may occasionally suffer mild-to-severe chronic hemolytic anemia. Chronic non-spherocytic hemolytic anemia (CNSHA) is a potential result of the Class I G6PD variants. This comparative computational study attempted to correct the defect in variants structure by docking the AG1 molecule to selected Class I G6PD variants [G6PDNashville (Arg393His), G6PDAlhambra (Val394Leu), and G6PDDurham (Lys238Arg)] at the dimer interface and structural NADP+ binding site. It was followed by an analysis of the enzyme conformations before and after binding to the AG1 molecule using the molecular dynamics simulation (MDS) approach, while the severity of CNSHA was determined via root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), hydrogen bonds, salt bridges, radius of gyration (Rg), solvent accessible surface area analysis (SASA), and principal component analysis (PCA). The results revealed that G6PDNashville (Arg393His) and G6PDDurham (Lys238Arg) had lost the direct contact with structural NADP+ and salt bridges at Glu419 - Arg427 and Glu206 - Lys407 were disrupted in all selected variants. Furthermore, the AG1 molecule re-stabilized the enzyme structure by restoring the missing interactions. Bioinformatics approaches were also used to conduct a detailed structural analysis of the G6PD enzyme at a molecular level to understand the implications of these variants toward enzyme function. Our findings suggest that despite the lack of treatment for G6PDD to date, AG1 remains a novel molecule that promotes activation in a variety of G6PD variants.

LEP G2548A polymorphism is associated with increased serum leptin and insulin resistance among T2DM Malaysian patients.

Background: Type 2 diabetes mellitus (T2DM) is a chronic metabolic syndrome that is rapidly increasing across the world, especially in Malaysia. Leptin plays a vital role in the regulation of metabolism through its effect on peripheral tissues. G2548A polymorphism in the LEP gene promoter has been associated with insulin resistance, leptin, and type 2 diabetes mellitus across different population, but has not been inclusively reported within the Malaysian population. Objective: Thus, our study aimed to investigate the impact of G2548A polymorphism on serum leptin levels and insulin resistance among Malaysian T2DM patients. Methods: This case-control study involved 150 T2DM patients and 150 non-diabetic volunteers from ethnic Malays, Chinese and Indians. Genotyping of G2548A polymorphism was carried out using PCR-RFLP. Serum leptin and insulin levels were determined via ELISA. ANOVA and Chi-square tests were used to determine the distribution of genotypes and allelic frequencies based on serum leptin and insulin levels. Results: Frequency of AA genotype and A allele of G2548A variant were significantly (P < 0.05) higher in T2DM patients of Malay and Indian ethnicities (4%, 35%, and 36%, 57%, respectively) as compared to the control groups (0%, 22%, and 18%, 35%, respectively). Fasting serum leptin levels were significantly (P < 0.001) higher in T2DM patients compared to non-diabetic subjects (166.78 pg/ml, 101.94 pg/ml, respectively). Additionally, elevated serum leptin, insulin levels, and BMI in diabetic patients were found to be associated with the AA genotype of this variant, compared to GG, and GA genotypes (P < 0.05). Conclusion: Our findings suggest a significant association between G2548A polymorphism among Malaysian T2DM subjects, particularly among Malay and Indian ethnic groups. Moreover, the A allele frequency of the G2548A variant significantly increased the risk of T2DM and is significantly associated with increased serum leptin, insulin levels, and elevated BMI.